Nature
would never leave something as valuable as genetic code unprotected. One
of the security measures protecting DNA in plants and animals is a mechanism
that fights viruses by shutting down their genes. In plants the phenomenon
is known as gene silencing, and it has been widely used to study plant
genes. It has also been used to create transgenic tomato plants that are
resistant to plant viruses.

Schematic presentation of the second step of
the RNAi pathway. View
larger

Courtesy H. Cargill

What works in tomatoes may also work in people. A number of laboratories
are investigating ways to exploit the naturally occurring defense mechanism
in humans to fight HIV, the virus that causes AIDS. A new study reports
success in blocking the virus in cell cultures by silencing genes in HIV
and human cells.

Philip A. Sharp, of the Massachusetts Institute of Technology in Cambridge,
and colleagues silenced the main structural protein in the virus, p24,
and the human protein CD4, which the virus needs to enter the cell. The
strategy impairs the virus in infected cells and limits its spread into
healthy cells. The study, co-led by Premlata Shankar of the Center for
Blood Research at Harvard Medical School in Boston, appears in Nature
Medicine.

"We were able to inhibit the production of the virus either by blocking
new infections or blocking the production of new viral particles in infected
cells," says Judy Lieberman, also of the Center for Blood Research.
"That's pretty encouraging."

The concept of silencing genes in HIV is straightforward: Hit the virus
where it counts by eliminating a protein it needs to reproduce or cause
infection. The human version of gene silencing has a different nameRNA
interference, or RNAibut the mechanism is fundamentally similar
to the one in plants and involves some of the same proteins.

In RNAi, short, interfering double-stranded RNA (siRNA) molecules are
added to the cell. The cell recognizes and degrades messenger RNA corresponding
to the target sequence. As a result, little or no protein is produced.

This is a kind of secret weapon inside us that we have not noticed
until now.

The key to RNAi is using strands shorter than about 30 base pairs, because
longer strands can cause the cell to commit suicide. "The cell realizes
it is infected by the virus and commits hara-kiri to prevent its spread
to neighbors," says Carl D. Novina, a member of the research team
at MIT.

This self-destruction is known as the interferon response; Sharp's team
bypassed the response by injecting siRNAs that were 21 to 23 base pairs
long.

What makes RNAi so exciting to many researchers is its potential for
knocking out a protein without harming the cell. By comparison, chemotherapy
invariably kills tumors by destroying cancerous cells as well as healthy
cells nearby.

"This method is extremely specific," says Lieberman. "You
can shut off HIV without interfering with any genes other than the ones
you target." Cells are basically missing 21 nucleotidesthe
RNA that is degradedand the rest of the genome is untouched.

The researchers tested the plausibility of pre-loading human cells with
siRNA as a way to protect against infection. They took latently infected
human cells, activated the HIV genes and were still able to block the
production of new viral particles. After entering a host cell, HIV inserts
itself into the genome, where it can reside without expressing its genes.

As with the drug 'cocktails' that patients take to kill HIV at different
stages of its life cycle, the most effective RNAi strategy will likely
include multiple targets. These could be targets that block entry into
the cell and disrupt the virus life cycle inside the cell.

The selection of targets is critical, and HIV mutates so rapidly that
a target RNA sequence may become obsolete in a short period of time. This
is one reason to silence genes in the host cell.

After identifying short RNA sequences in the target proteins, the researchers
screen the sequences against the entire human genome, a process that takes
about twenty minutes on a computer. "The critical thing in selecting
targets is to chose unique RNA sequences that do not correspond to genes
required for normal cell metabolism," says Novina.

"This is just the beginning of a wave of studies on using siRNA
to inhibit viral infection," says Thomas Tuschl, of the Max-Planck-Institute
for Biophysical Chemistry in Göttingen, Germany. "It's clear
that siRNA is basic research at the moment, but the technology can now
be evaluated as a potential therapy."

Even if no therapies are developed using RNAi, the technology will help
researchers dissect the biology of HIV infection and design drugs based
on the information.

"You can turn off a human gene for a week and then challenge the
cell with HIV to see whether the cell gets infected," says Tuschl.
"This will help us understand how HIV functions in the cell and will
have a big impact on developing new drugs."

"The next big breakthrough will be when we learn that injecting
siRNA into an organism does the same thing as it does in tissue cultures,"
he says, noting that such work will probably happen in mice.

Tuschl led the study last year that helped inspire Novina and Lieberman
to test RNAi against HIV. His team reported in Nature that short
strands could 'sneak in under the radar' and trigger a silencing mechanism
in human kidney cells and other mammalian cells.

The effects of adding siRNA to cells are transient, and finding ways
to deliver siRNA to human cellsor engineer cells to express themremains
a primary challenge for the field.

Researchers at the City of Hope Cancer Center in Duarte, California,
recently developed a DNA-based delivery system. As reported in Nature
Biotechnology, they generated human cells that produced siRNA against
the REV protein, which is important in causing human disease.

"Our goal is to engineer human cells to be resistant against attack
by the virus and also make them incapable of spreading HIV," says
John Rossi, who led the study. The idea is that patients could one day
be given a population of modified cells that are highly resistant to viral
infection.

"As with all technological advances, it remains to be seen how generally
useful it will be," says Rossi, noting that RNAi "is not the
universal panacea for antiviral therapy." He points out that some
viruses have proteins that allow them to circumvent the mechanism.

The purpose of RNAi in animals has not yet been demonstrated, but some
clues to its role were reported recently by scientists at the University
of California, Riverside, who study the flock house virusan animal
virus that can infect plants. The researchers discovered that the flock
house virus contains a protein that suppresses gene silencing in both
plants and flies.

The fact that one suppressor protein works in two kingdoms is strong
evidence that the silencing pathways in plants and animals are related,
the team concludes in Science. Furthermore, the finding suggests
that animal cells have used RNAi to protect themselves from viral attacks
in a similar manner as plants.

"We show that RNAi is a natural antiviral defense," says Shou-Wei
Ding, who led the study. "This is a kind of secret weapon inside
us that we have not noticed until now. And one reason we haven't noticed
it is that viruses contain proteins that suppress the weapon."